Project Summary Hepatocyte nuclear factor 4 (HNF4) is a highly conserved member of the nuclear receptor (NR) superfamily of ligand-dependent transcription factors. It is an essential gene, playing a critical role in early development, as well as in the adult in the liver, kidney, pancreas and intestine. HNF4 has been directly linked to several human diseases including diabetes and hemophilia and indirectly linked to others including atherosclerosis and cancer. HNF4 is one of the most abundant transcription factors in the liver. Whereas many target genes have been identified for HNF4, recent genome-scale analyses indicate that there are many more yet to be identified. Recent results indicate, for example, that there may be differences in the target genes of the different isoforms of HNF4 generated by alternative splicing and promoter usage. Furthermore, HNF4 is known to be a heavily phosphorylated protein and to respond to a variety of intra- and extracellular signals; however, only a few of the phosphosites have been mapped and fully characterized. Finally, in addition to its role in intermediary metabolism, there is a growing body of evidence indicating that HNF4 may also play a role in regulating the cell cycle. In order to address these issues, we propose the following three Specific Aims: 1) Identify new target genes for HNF4 using genome-scale analysis and an in vivo mouse model to examine the functional differences in HNF4 isoforms; 2) Investigate the role of tyrosine phosphorylation in HNF4 function; and 3) Investigate the role of HNF4 in regulating the cell cycle. The proposed experiments will continue the investigation of the role of HNF4 in liver-specific gene expression using a broad array of modern techniques. The results will further our understanding of the mechanisms of tissue-specific gene regulation. They will also provide invaluable information regarding a variety of human diseases that are linked to HNF4. Finally, as a potential drug target, a more comprehensive knowledge of the genes targeted by HNF4, as well as the signaling pathways that target HNF4, will be essential to developing appropriate therapies. Project Narrative Our research is relevant to public health because it attempts to decipher the mechanisms by which genes are turned on in the liver and gut. This is critical not only for understanding the causes of diseases such as diabetes, obesity, atherosclerosis and cancer, but also for designing new therapies to treat those diseases.